E4orf1 improves adipose tissue-specific metabolic risk factors and indicators of cognition function in a mouse model of Alzheimer’s disease

Objective Obesity, impaired glycemic control, and hepatic steatosis often coexist and are risk factors for developing dementia, and Alzheimer’s disease (AD). We hypothesized that a therapeutic agent that improves glycemic control and steatosis may attenuate obesity-associated progression of dementia. We previously identified that adenoviral protein E4orf1 improves glycemic control and reduces hepatic steatosis despite obesity in mice. Here, we determined if this metabolic improvement by E4orf1 will ameliorate cognitive decline in a transgenic mouse model of AD. Methods Fourteen- to twenty-month-old APP/PS1/E4orf1 and APP/PS1 (control) mice were fed a high-fat diet. Cognition was determined by Morris Water Maze (MWM). Systemic glycemic control and metabolic signaling changes in adipose tissue, liver, and brain were determined. Results Compared to control, E4orf1 expression significantly improved glucose clearance, reduced endogenous insulin requirement and lowered body-fat, enhanced glucose and lipid metabolism in adipose tissue, and reduced de novo lipogenesis in the liver. In the brain, E4orf1 mice displayed significantly greater expression of genes involved in neurogenesis and amyloid-beta degradation and performed better in MWM testing. Conclusion This study opens-up the possibility of addressing glycemic control and steatosis for attenuating obesity-related cognitive decline. It also underscores the potential of E4orf1 for the purpose, which needs further investigations.


Results
E4orf1 protein is expressed only in the adipose tissue of APP/PS1/E4 mice.
To confirm transgenic expression of E4orf1 protein specific to the adipose tissue, protein lysates from ingunal (subcutaneous), liver, hippocampus and cortex were immunoblotted with E4orf1 antibody. As seen in Fig. S1, western blot analysis showed E4orf1 protein expression only in the ingunal (iWAT) adipose tissue depot of APP/PS1/E4 and not in APP/PS1 mice. E4orf1 protein expression was not observed in the liver, hippocampus or cortex.

Real-Time Quantitative PCR
Total RNA was extracted from the adipose tissue, liver and brain tissue using RNeasy® Plus The expression level of genes associated with adipose tissue, liver and brain tissue lipid metabolism, mitochondrial function and inflammation were determined by quantitative real-time polymerase chain reaction (qRT-PCR). Specific primers for each gene were designed using Sigma Aldrich Oligo Architect software, listed in Supplemental Table S1. The RT-PCR reaction mix had a final volume of 20 µL; 50 or 25 ng of cDNA, 450 nM of the forward and reverse primers, and 10 µL of 1X SsoAdvanced™ Universal SYBR® Green Supermix (Bio-Rad Laboratories, cat. no. 172-5271). PCR reactions were carried out in 96-well plates using the Bio-Rad CFX RT-PCR detection system. All reactions were performed in duplicates. Mouse B2m and GAPDH genes were used as the reference.

Western Blotting
Protein lysate was extracted from the inguinal adipose tissue depot, liver and brain tissue by lysing in modified radioimmunoprecipitation assay buffer (RIPA buffer; 10X RIPA buffer from Cell Signaling (Cat no: 9806). 1X RIPA Buffer: 20 mM Tris-HCl (pH 7.5) 150 mM NaCl, 1 mM Na2 EDTA 1 mM EGTA 1% NP-40 1% sodium deoxycholate 2.5 mM sodium pyrophosphate 1 mM b-glycerophosphate 1 mM Na3 VO4 1 µg/ml leupeptin) as previously described. Protein extracts were separated using SDS-PAGE gel (Bio-Rad, Hercules, CA) and transferred on to a nitrocellulose membrane using Turbo transfer system (Bio-Rad, Hercules, CA). The nitrocellulose membrane was blocked using 10% non-fat milk in TBST (We utilized VWR's 20X liquid ultrapure TBS Buffer (catalog number J640) and added a 1:100 ratio of Tween 20, using a 1X dilution for washing the western blot. We also used as a dilution buffer for the antibodies.

Morris water maze (MWM) for spatial memory testing
The spatial learning and memory capabilities of the mice were evaluated with MWM test (2). The test tank was 120 cm in diameter and 75 cm deep with visual cues placed around. The water tank was divided into four quadrants, north-west (NW), north-east (NE), south-west (SW), and southeast (SE). A circular opaque platform (36 cm in diameter) was located at the SW quadrant about 2 cm below the water level. A platform marker or 'flag' was mounted at the top of the platform to provide a visual cue to the position of the platform. The tank was filled with clear water, and the temperature was always maintained at 25-26 o C using an electric heater. The testing procedures were divided into three major phases: training phase, testing phase, and spatial probe test phase.

Phase 1 (training phase):
During the training phase, the mice underwent three training sessions each day for two days. The mice were placed into the water from a randomly selected starting position away from the fixed platform. This position was the same for all mice during that trial.
The escape latency was set for 60 seconds, and after each trial, mice were placed on the platform for 20 seconds irrespective of if they reached or not. The time for escape latency was recorded using a stopwatch.

Phase 2 (testing phase):
The testing session was performed for 2 consecutive days (Trial 1 and 2), and 2 additional days (Trial3 and 4) after a gap of 7 days. Once again, the platform was in the fixed position located at the SW quadrant. The starting position of the mice being placed in the tank was different at each trial. Each mouse had 3 trials per day for each trial, therefore each mouse went through 12 trials over the 4 days. The water was camouflaged to appear opaque using liquid tempera paint in the tank. The platform flag was removed during testing and the mice movement was video recorded.

Phase 3 (spatial probe test):
After, the mice underwent a one-time 60-second probe trial. The water was kept opaque, the platform removed, and the mice placed at a novel starting position. The